The present invention relates to antibodies, particularly monoclonal antibodies which recognise particular epitopes of the intimin protein of enteropathogenic E. coli and enterohemorrhagic E. coli, their use in the detection of such enterophathogenic E. coli, and kits containing such antibody for such use.
Enteropathogenic Escherichia coli (EPEC) were the first E. coli linked with diarrhoea in humans. They are a sub-group of pathogenic E. coli that are a major cause of infant diarrhoea. Enterohemorrhagic E. coli (EHEC) are another such sub-group. EPEC normally fall into several distinct serotype groups based on the type of lipopolysaccharide which is expressed at their cell surface. The most common serogroups that are associated with EPEC are O26, O55, O111, O114, O126, O127, O128, O142 and O157. Although EPEC and EHEC infections are still a serious problem world-wide, no specific treatment or vaccine is as yet available for treating these diseases and furthermore, no relatively simply and/or accurate tests are as yet available for diagnosis.
One major problem in diagnosing EPEC infections is that, apart from the link with several O serotypes, EPEC are difficult to distinguish from commensal, non-pathogenic E. coli which are usually resident in the intestines of healthy individuals. Ultra-structural studies of intestinal biopsy specimens from children with EPEC-induced diarrhoea have shown that EPEC attach to the intestinal epithelium in a characteristic fashion, which is central to the pathogenesis of the disease.
Wherever bacteria bind to the brush border of the intestine, microvilli are destroyed, or xe2x80x9ceffacedxe2x80x9d. The apical cell membrane of the bare enterocytes form cup-like pedestals upon which the bacteria intimately adhere. EHEC, Hafnia alvei and Citrobacter freundii biotype 4280 have also been shown to induce similar lesions, termed effacement and attachment lesions, to the intestinal brush border of infected hosts.
Pedestal formation is associated with accumulation of polymerised actin below the surface of the eukaryotic cell directly engulfing the bacteria. This polymerised actin can be detected by staining with a specific dye called phalloidin. A diagnostic test, based on the use of this stain with cultured mammalian cells incubated with pathogenic bacteria has been developed. However, this test is not EPEC-specific, and is expensive and time consuming, which renders its routine use difficult.
The EPEC or EHEC gene product that mediates intimate attachment to epithelial cells is a protein called intimin (IntEPEC and IntEHEC respectively), which is a 94 kdal outer membrane protein encoded by the chromosomal eaeA gene. The amino-terminis of IntEPEC and IntEHEC have a high degree of homology with the eae gene products of Citrobacter freundii biotype 4280 (IntCF) and also with the amino terminus of invasin (Inv), the product of inv gene of Yersinia pseudotuberculosis (InvYP) and Yersinia enterocolitica (InvYE).
The DNA and protein sequences of various intimins were disclosed by Kaper (Proc. Natl. Acad. Sci. USA, 87: 7839-7843 (1990) and Mol. Microbiol., 6: 411-417 (1992)). However, the proteins themselves were not purified. Frankel et al. (Infec. Immun., 62: 1835-1842 (1994)) have isolated the intimin proteins from various sources and have generated Maltose-binding protein (MBP), fusion proteins carrying particular domains of the intimin proteins.
The cell binding activity of InvYP resides predominantly in the 192 carboxy-terminus amino acids of the protein. Previously, Frankel et al. (supra) used fusions to maltose binding protein (MBP) to show that, like Inv, an Int domain which mediates receptor binding resides in the 280 (or 275 in the case of EHEC) amino acids of the carboxy-terminus of the protein (IntEPEC280, IntEHEC275, IntHA280, IntCF280).
However, unlike Inv, which by itself can convert an E. coli K12 strain to an organism capable of attaching and then invading mammalian cells, Int requires the cooperation of other EPEC proteins to induce the effacement and attachment lesion. Notwithstanding that, however, IntEPEC is a virulent factor that induces seroconversion in human volunteers (Donnenberg et al, J. Clin. Invest., 92: 1412-1417 (1993)). The carboxy-terminal domain of this protein is able to bind to target cells and thus it is likely to be surface exposed, thus making it accessible, on whole bacteria, to immune reagents.
We have now generated further fusion proteins in addition to MBP-IntEPEC280 (amino acids 660-939). These fusion proteins include different amino acid sequences derived from the 280 binding domain. MBP-IntEPEC150 (amino acids 790-939) was found to be the smallest fusion protein that had the ability to mediate cell binding. In addition the cysteine residue at position 937 of Int seems to be required for binding activity, as substitution with serine results in a loss in biological activity. Using the MBP expression system we have obtained high yields of MBPIntEPEC280, MBP-IntEHEC275, MBP-IntCF280 and MBP-InvYP280 fusion proteins. These proteins can be used to immunise mice in order to generate specific monoclonal antibodies. We have now identified monoclonal antibodies which specifically recognise the antigen MBP-IntEPEC280 and which do not recognise MBP, MBP-IntEHEC275, MBP-IntCF280 or MBP-InvYP280. These antibodies are therefore specific for the 280 amino acid domain of IntEPEC and will therefore be useful in both the detection and/or treatment of EPEC infection.
Similarly, MBP-IntEHEC275 can be used to generate monclonal antibodies which recognise MBP-IntEHEC275 but not MBP-IntEPEC280.
Thus, in a first aspect, the present invention provides an antibody which recognises a region within either the carboxy-terminus 280 amino acid domain of the enteropathogenic E. coli intimin protein (IntEPEC280) or the enterohemorrhagic E. coli intimin protein (IntEPEC280).
In the context of the present invention, xe2x80x9cantibodyxe2x80x9d refers to conventional polyclonal and monoclonal antibodies and includes antigen binding portions or fragments thereof, e.g. Fab and/or fragments of Fab.
In a preferred embodiment, the antibody is a monoclonal antibody. In particularly preferred embodiments, the antibody is a monoclonal antibody which recognises a region within the following carboxy-terminus 280 amino acid sequence:
or
a region within the following carboxy-terminus 275 amino acid sequence:
Preferably, a monoclonal antibody recognizing IntEPEC280 does not recognise the carboxy-terminus 275 amino acid domain of the intimin protein from enterohemorragic E. coli (IntEHEC275) or the carboxy-terminus 280 amino a id domain of Citrobacter freundii biotype 4280 IntCF280 or the invasin protein from Yersinia pseudotuberculosis (InvYP280).
More preferably, the monoclonal antibody recognising IntEPEC280 recognises an epitope in the 660-790 amino acid region of the enterophathogenic E. coli intimin protein.
Hybridomas producing monoclonal antibodies of the invention are also included within the scope of the invention.
The properties of the monoclonal antibodies of the invention render them useful for detecting enterophathogenic E. coli and enterohemorrhagic E. coli. Thus, the invention also provides the use of such monoclonal antibodies in detecting enteropathogenic and enterohemorrhagic E. coli. 
In a further aspect, the invention provides a method for detecting the presence of enteropathogenic or enterohemorrhagic E. coli which comprises the step of bringing a sample to be tested into contact with a monoclonal antibody of the invention. In one embodiment, the monoclonal antibody of the invention could be attached to a solid support, for example a test strip or a microtitre well, and the sample containing the antigen could be brought into contact with it.
Suitably, the method will be an ELISA method.
In a further aspect, the invention provides a method for detecting enteropathogenic or enterohemorrhagic E. coli infection in a subject which comprises the step of contacting a monoclonal antibody of the invention with a sample obtained from the subject. Suitably, the sample will be a faecal sample.
The fusion proteins (MBP-IntEPEC280 and MBP-IntEHEC275) are particularly convenient for generating hybridomas capable of producing monoclonal antibodies of the invention, and form another aspect of the present invention.
In a final aspect, the invention provides a peptide having the sequence:
The invention will now be described with reference to the following examples which should not be construed as in any way limiting the invention.